The goal of the project is to study the mechanisms by which vascular endothelium is damaged during inflammatory reactions. The major focus has been on endothelial cell damage induced by the macrophage and macrophage products using apoptosis (programmed cell death) as a measure of damage. Coculture of human umbilical vein endothelial cells (HUVEC) or bovine aorta endothelial cells (BAEC) with several different macrophage-like cell lines have shown that BAEC are much more sensitive to damage than are HUVEC. The differential sensitivity to macrophage induced damage is paralleled by an increased sensitivity to products released from the macrophage during activation. Tumor necrosis factor and nitric oxide by themselves will induce apoptosis in BAEC with only a minimal effect on HUVEC. Studies on the release of reactive oxygen species, superoxide and hydrogen peroxide, from macrophages in parallel with measurements of apoptosis demonstrate that reactive oxygen species do not contribute to macrophage induced apoptosis in BAEC or HUVEC. In a variety of vasculitic diseases and in systemic lupus erythematosus, anti-endothelial cell antibodies are present in patient serum and are hypothesized to contribute to disease pathogenesis. Little is known, however, about the mechanisms by which they contribute to coronary artery disease and thrombosis. Binding of antibodies specifically to apoptotic endothelial cells could result in opsonized particles capable of initiating macrophage activiation and release of pro-inflammatory mediators. To test this hypothesis, methods for measuring surface antigen expression on apoptotic endothelial cells using flow cytometry have been developed. This methodology will be used to screen sera from lupus patients for antibodies specific for apoptotic HUVEC. Preliminary studies have been initiated with Dr. Alayash to develop an in vitro model system for assessing hemoglobin damage of endothelium and the role of reactive oxygen and nitrogen species as mediators of damage.